Multicolor luciferase reporter assays are powerful tools for simultaneously monitoring the expression of several genes. Dual-color luciferase reporter assays were established in 2004 and have been used in broad range of application from basic researches to applications in clinical research, e.g., drug screening and alternative toxicological tests. The advantage of this method is that it can be used to simultaneously monitor sequential and complex changes in the expression of multiple genes from the single-cell level to the tissue level. In this review, I focus on the simultaneous analysis of multiple genes using multicolor beetle luciferases.